Bottom Line:
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain.ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix.This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase.

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy.

ABSTRACTUsing two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

fig1: ICAP-1 inhibition of cell spreading and rescue by V12Cdc42. Cells were transiently transfected, detached with EDTA, plated on fibronectin-coated coverslips (5 Î¼g/ml) in serum-free medium, fixed, and stained with different antibodies. (A) NIH3T3 cells transfected with mycâ€“ICAP-1 and plated on fibronectin for 1 h (a) and for 24 h (b). NIH3T3 cotransfected with mycâ€“ICAP-1 and myc-V12Cdc42 plated for 1 h on fibronectin (c). V12Cdc42-NIH3T3 transiently transfected with mycâ€“ICAP-1 and plated for 1 h on fibronectin (d). Transfected cells were visualized by anti-myc monoclonal antibody (left), whereas actin organization was detected in the same sample with FITC phalloidin (right). (B) The percentage of spread cells was calculated as described in Materials and methods. Values are means Â± SD from three independent experiments in which >20 cells per condition were scored.

Mentions:
To test the possible function of ICAP-1, we transiently transfected this molecule in NIH3T3 cells. As detected with our polyclonal antibody, the endogenous level of ICAP-1 was very low in these cells and raised âˆ¼20 times after transfection. Transfected cells were detached from the dish and replated on fibronectin-coated glass to test their ability to adhere and spread. As shown in Fig. 1 , cells expressing ICAP-1 attach to fibronectin, but only 35% Â± 6 spread after 60 min of plating on the coated substratum, whereas 84% Â± 4 of control untransfected cells present in the same sample spread under the same plating conditions. The inhibition of cell spreading by ICAP-1 expression was not the result of a toxic or non specific effect, since cells recover from inhibition and spread normally after overnight incubation on fibronectin (Fig. 1 A, b). Moreover, expression of a different Î²1 integrin interactor, such as melusin (Brancaccio et al., 1999), did not alter cell adhesion or spreading (unpublished data).

fig1: ICAP-1 inhibition of cell spreading and rescue by V12Cdc42. Cells were transiently transfected, detached with EDTA, plated on fibronectin-coated coverslips (5 Î¼g/ml) in serum-free medium, fixed, and stained with different antibodies. (A) NIH3T3 cells transfected with mycâ€“ICAP-1 and plated on fibronectin for 1 h (a) and for 24 h (b). NIH3T3 cotransfected with mycâ€“ICAP-1 and myc-V12Cdc42 plated for 1 h on fibronectin (c). V12Cdc42-NIH3T3 transiently transfected with mycâ€“ICAP-1 and plated for 1 h on fibronectin (d). Transfected cells were visualized by anti-myc monoclonal antibody (left), whereas actin organization was detected in the same sample with FITC phalloidin (right). (B) The percentage of spread cells was calculated as described in Materials and methods. Values are means Â± SD from three independent experiments in which >20 cells per condition were scored.

Mentions:
To test the possible function of ICAP-1, we transiently transfected this molecule in NIH3T3 cells. As detected with our polyclonal antibody, the endogenous level of ICAP-1 was very low in these cells and raised âˆ¼20 times after transfection. Transfected cells were detached from the dish and replated on fibronectin-coated glass to test their ability to adhere and spread. As shown in Fig. 1 , cells expressing ICAP-1 attach to fibronectin, but only 35% Â± 6 spread after 60 min of plating on the coated substratum, whereas 84% Â± 4 of control untransfected cells present in the same sample spread under the same plating conditions. The inhibition of cell spreading by ICAP-1 expression was not the result of a toxic or non specific effect, since cells recover from inhibition and spread normally after overnight incubation on fibronectin (Fig. 1 A, b). Moreover, expression of a different Î²1 integrin interactor, such as melusin (Brancaccio et al., 1999), did not alter cell adhesion or spreading (unpublished data).

Bottom Line:
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain.ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix.This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase.

Affiliation:
Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy.

ABSTRACTUsing two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.